Project Summary Myelination of axons in the central nervous system (CNS) is required for the propagation of electrical activity in neurons and maintenance of axonal health. Oligodendrocyte progenitor cells (OPCs) give rise to the myelinating cells of the CNS, known as oligodendrocytes. While the majority of OPCs differentiate into oligodendrocytes, a population of OPCs remains undifferentiated and evenly distributed in the CNS throughout life. How OPCs space during development and maintain their spacing into adulthood is unknown. In vivo imaging in adult mouse cortex and the spinal cord of zebrafish larvae demonstrate that OPCs undergo a process termed tiling. We define tiling as the dynamic process that OPCs undergo during development where they establish and maintain even spacing and distinct territories that, under non-pathological conditions, do not overlap. During tiling, OPCs also exhibit contact-mediated repulsion (CMR). CMR is a process whereby migrating OPCs will retract their processes and change their migratory direction following contact with other OPCs. However, the molecular mechanisms that OPCs use to facilitate tiling and CMR are unknown. Additionally, demyelinating diseases cause increased OPC clustering, which signifies a disruption in local tiling mechanisms. Understanding the fundamental mechanisms of that regulate OPC-OPC interactions will provide insight into the developmental processes of OPC tiling and potential targets for modulating OPC migration and recruitment to demyelinated lesions. The purpose of this proposal is to elucidate the mechanisms that facilitate the rapid tiling of OPCs during development by investigating three important processes during tiling: (1) OPC proliferation, (2) OPC migration, and (3) OPC CMR. To investigate OPC tiling during development, I will use zebrafish (Danio rerio) as a vertebrate model system. The relative simplicity of this system and clarity of the spinal cord of zebrafish embryos and larvae allow for visualization of OPC migration, proliferation, and CMR in a large portion of the developing CNS. I will use a candidate gene approach based on previous literature to identify potential mediators of proliferation and CMR. Additionally, I will investigate candidates identified from an unbiased small molecule screen to identify novel mediators of OPC migration during developmental tiling. I will then use a combination of CRISPR/Cas9 gene editing techniques, pharmacological inhibitors, in vivo imaging, and immunohistochemistry to characterize the role of OPC proliferation, migration, and CMR to developmental OPC tiling. Overall, this work will expand our understanding of OPC tiling and interactions and potentially provide translational targets for demyelinating diseases, such as multiple sclerosis.